AMP-protein activated Kinase (AMPK) is a sensor and regulator of cellular energy homeostasis, is a master switch regulating glucose and lipid metabolism and activation of AMPK results in many beneficial effects. (Misra, et al, The role of AMP kinase in diabetes, Indian J Med Res 125:389-398 (2007); Kola, et al, The Role of AMP-Activated Protein Kinase in Obesity, Obesity and Metabolism, Vol 36 (2008); Kahn, et al, AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 1(10:15-25 (2005); Hardie, D. G. and Hawley, S. A. AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 23: 1112 (2001), Kemp, B. E. et.al. AMP-activated protein kinase, super metabolic regulator. Biochem. Soc. Transactions 31:162 (2003)). AMPK can be activated by three distinct mechanisms; 1) allosteric activation, 2) stimulation of phosphorylation of the alpha-subunit on Thr172 by upstream kinase(s), and 3) inhibition of dephosphorylation by protein phosphatases (Kola, et al, The Role of AMP-Activated Protein Kinase in Obesity. Obesity and Metabolism 36:198-211 (2008)). The resulting activation leads to a decrease in fatty acid synthesis and oxidation, and a decrease in cholesterol synthesis (Carling, D. et.al. A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Letters 223:217 (1987)). Other effects of AMPK activation include positive changes in the levels of potential drug targets for components of the metabolic syndrome including hormone sensitive lipase, glycerol-3-phosphate acyltransferase, malonyl-CoA decarboxylase, and hepatocyte nuclear factor-4.alpha. AMPK activation stimulates glucose transport in skeletal muscle and controls expressional regulation of key genes in fatty acid and glucose metabolism in liver (summarized in U.S. patent application Ser. No. 7,119,205).
Genomic pathways that AMPK activation affects include decreased expression of glucose-6-phosphatase (a key enzyme in hepatic glucose production) (Lochhead, P. A. et.al. 5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase. Diabetes 49:896 (2000)), and SREBP-1c (Zhou, G. et.al. Role of AMP-activated protein kinase in mechanism of metformin action. The J. of Clin. Invest. 108: 1167 (2001)), a key lipogenic transcription factor. Note that the metabolic changes induced by AMPK activation are both acute changes due to phosphorylation of key enzymes, and longer-term effects on the expression of genes involved in metabolic regulation.
There have been several studies that indicate that activation of AMPK will result in many benefits. In the liver, there is decreased glucose output and improvement in glucose homeostasis, decreased fatty acid and cholesterol synthesis and increased fatty acid oxidation. In skeletal muscle tissue there is increased glucose uptake and fatty acid oxidation. There is a reduction in intra-myocyte triglyceride accumulation and improved insulin action. A reduction in the ability to store fat, due to the down-regulation of fatty acid synthesis, results in long-term weight reductions. The combinations of all these effects are an excellent treatment for metabolic syndrome, diabetes and obesity.
Several studies in rodents and humans support that AMPK activation leads to substantial benefits (Bergeron, R. et.al. Effect of 5-aminoimidazole-4-carboxamide-1(beta)-D-ribofuranoside infusion on in vivo glucose metabolism in lean and obese Zucker rats. Diabetes 50:1076 (2001), Song, S. M. et.al. 5-Aminoimidazole-4-darboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabeted (ob/ob) mice. Diabetologia 45:56 (2002), Halseth, A. E. et.al. Acute and chronic treatment of ob/ob and db/db mice with AICAR decreases blood glucose concentrations. Biochem. and Biophys. Res. Comm. 294:798 (2002), Buhl, E. S. et.al. Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying feature of the insulin resistance syndrome. Diabetes 51: 2199 (2002)). Activation of AMPK increases mitochondrial biogenesis (Reznick, et al, The role of AMP-activated protein kinase in mitochondrial biogeneses. J Physiol 574.1 (2006)). Reduced mitochondria content is important in the pathogenesis of insulin resistance and type 2 diabetes.
Many in vivo studies have relied on the AMPK activator 5-Aminoimidazole-4-darboxamide ribonucleoside (AICAR), a cell permeable precursor of ZMP. ZMP acts as an intracellular AMP mimic, and, when accumulated to high enough levels, is able to stimulate AMPK activity (Corton, J. M. et.al. 5-Aminoimidazole-4-carboxamide ribonucleoside, a specific method for activating AMP-activated protein kinase in intact cells? Eur. J. Biochem. 229: 558 (1995)). Several in vivo studies have demonstrated beneficial effects of both acute and chronic AICAR administration in rodent models of obesity and type 2 diabetes (Bergeron, R. et.al. Effect of 5-aminoimidazole-4-carboxamide-1(beta)-D-ribofuranoside infusion on in vivo glucose metabolism in lean and obese Zucker rats. Diabetes 50:1076 (2001), Song, S. M. et.al. 5-Aminoimidazole-4-darboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabeted (ob/ob) mice. Diabetologia 45:56 (2002), Halseth, A. E. et.al. Acute and chronic treatment of ob/ob and db/db mice with AICAR decreases blood glucose concentrations. Biochem. and Biophys. Res. Comm. 294:798 (2002), Buhl, E. S. et.al. Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying feature of the insulin resistance syndrome. Diabetes 51: 2199 (2002)). For example, 7 week AICAR administration in the obese Zucker (fa/fa) rat leads to a reduction in plasma triglycerides and free fatty acids, an increase in HDL cholesterol, and a normalization of glucose metabolism as assessed by an oral glucose tolerance test (Minokoshi, Y. et.al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415: 339 (2002)). In both ob/ob and db/db mice, 8 day AICAR administration reduces blood glucose by 35% (Halseth, A. E. et.al. Acute and chronic treatment of ob/ob and db/db mice with AICAR decreases blood glucose concentrations. Biochem. and Biophys. Res. Comm. 294:798 (2002)). In addition to AICAR, more recently it was found that the diabetes drug metformin can activate AMPK in vivo at high concentrations (Zhou, G. et.al. Role of AMP-activated protein kinase in mechanism of metformin action. The J. of Clin. Invest. 108: 1167 (2001), Musi, N. et.al. Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes 51: 2074 (2002)).
In addition to pharmacologic intervention, several transgenic mouse models have been developed in the last years, and initial results are becoming available. Expression of dominant negative AMPK in skeletal muscle of transgenic mice has demonstrated that the AICAR effect on stimulation of glucose transport is dependent on AMPK activation (Mu, J. et.al. A role for AMP-activated protein kinase in contraction and hypoxia-regulated glucose transport in skeletal muscle. Molecular Cell 7: 1085 (2001)).
Lowering of blood pressure has been reported to be a consequence of AMPK activation (Buhl, E. S. et.al. Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying feature of the insulin resistance syndrome. Diabetes 51: 2199 (2002)), therefore activation of AMPK has beneficial effects in hypertension. Through combination of some or all of the above-mentioned effects stimulation of AMPK is expected to reduce the incidence of cardiovascular diseases (e.g. MI, stroke). Endothelial NO synthase (eNOS) has been shown to be activated through AMPK mediated phosphorylation (Chen, Z.-P., et.al. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Letters 443: 285 (1999)), therefore AMPK activation by any means can be used to improve local circulatory systems.
Increased fatty acid synthesis is a characteristic of many tumor cells, therefore decreased synthesis of fatty acids through activation of AMPK can be useful as a cancer therapy. Cell cultures exposed to AICAR to activate AMPK attenuated the growth of MDA-MB-231 tumors in nude mice (Swinnen, JV, et al. Mimicry of a Cellular Low Energy Status Blocks Tumor Cell Anabolism and Suppresses the Malignant Phenotype. Cancer Res 2005; 65:6 (2005)). The AMPK activator metformin inhibits breast cancer cells, pancreatic cancer (Zakikhani, et. at, Metformin is an AMP-Kinase-Dependent Growth Inhibitor for Breast Cancer Cells. Cancer Research 66, 10269-10273 (2006), (Schhneider, et. al, Metformin clearly inhibits pancreatic cancer. Cancer Detection and Prevention Online, Abstract 260 (2002)) and reduces overall cancer risk (Evans, et al, Metformin and reduced risk of cancer in diabetic patients. BMJ 330:1304-1305 (2005)). Resveratrol also activates AMPK to kill cancer cells (Hwang, et. al. Resveratrol Induces Apoptosis in Chemoresistant Cancer Cells via Modulation of AMPK Signaling Pathway. Annals of the New York Academy of Sciences, V 1095 Signal Transduction Pathways, Part C: Cell Signaling in Health and Disease, 441-448 (2007)).
There are several current methods to activate AMPK, but these methods have some problems associated with them. As stated above, AMPK can be activated with metformin, resveratrol and AICAR. But metformin may also produce lactic acidosis, which can become a life-threatening condition, especially where a patient has renal insufficiency. Still further, metformin therapy is often counter-indicated where a patient takes other drugs that interfere with renal function. Resveratrol has a very low bioavailability, and large amounts may be necessary in order to achieve efficacy. AICAR is banned by the World Anti-Doping Code for athletic events (The Prohibited List 2009, World Anti-Doping Agency, http://www.wada-ama.org/rtecontent/document/2009_Prohibited_List_ENG_Final_20_Sept_08.pdf), and is currently not available for human use.
Other methods to activate AMPK include diabetic drug class thiazolidinediones (LeBrasseur, et al, Thiazolidinediones can rapidly activate AMP-activated protein kinase (AMPK) in mammalian tissues. Am J Physiol Endocrinol Metab (2006) and (Matejkova, et al, Possible involvement of AMP-activated protein kinase in obesity resistance induced by respiratory uncoupling in white fat. FEBS Letters, 539(1-3):245-248 (2003)). However, various thiazolidinediones have been withdrawn from the market or development has discontinued due to relatively high hepatotoxicity. (Isley, Hepatotoxicity of thiazolidinediones. Expert Opinion on Drug Safety, 2(6):581-586 (2003)). Recently, overstimulation of PPAR gamma has also been implicated in increased chances of developing colorectal cancer. The withdrawal of troglitazone has led to concerns of the other thiazolidinediones also increasing the incidence of hepatitis and potential liver failure, an approximately 1 in 20,000 individual occurrence with troglitazone. Because of this, the FDA recommends two to three month checks of liver enzymes for the first year of thiazolidinedione therapy to check for this rare but potentially catastrophic complication. In addition, thiazolidinediones have a side effect of water retention, leading to edema. Recent studies have shown there may be an increased risk of coronary heart disease and heart attacks with the thiazolidinedione rosiglitazone (Clinical Trials for Rosiglitazone, www.ClinicalTrials.gov).
The reduction of calories below baseline ad. Librium feeding levels (Calorie Restriction) is one method to induce AMPK activation. The extension of lifespan in the nematode worm Caenorhabditis elegans are shown to be completely dependent on the activation (phosphorylation) of AMP-protein activated kinase (AMPK) and the FOXO transcription factor DAF-16 (Greer, et al, An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans. Current Biology 9:17(19):1646-56 (2007)). Under Calorie Restriction, cellular energy depletion causes rising AMP levels, and an increase in the Nicotinamide Adenine Dinucleotide (NAD+) level as compared to the reduced level (NADH), results in activation of AMPK (Raphaloff-Phail, et al Biochemical regulation of mammalian AMP-activated protein kinase (AMPK) activity by NAD and NADH. Journal of Biological Chemistry, Manuscript M409574200 (2004)). While Calorie Restriction is a low-risk method to activate AMPK, it requires the reduction of baseline food consumption to levels that are not desirable to the majority of the population due to extreme hunger. The diet is extremely difficult to follow over time.
Strenuous exercise can also activate AMPK (Lee-Young, et al “AMPK activation is fiber type specific in human skeletal muscle: effects of exercise and short-term exercise training, Journal of Applied Physiology, 2009 July; 107(1); 283-9) however, again, this may not be desirable or achievable by the majority of the population.
In animal models, various stresses such as oxidative stress, hypoxia, ischemia and heat shock can activate AMPK (Towler, et. al, AMP-activated protein kinase in metabolic control and insulin signaling Circ Res. 100, 328-341 (2007). These stresses are not recommended as an AMPK activation agent.
Undoubtedly, activation of AMPK can aid in the prevention or treatment of disorders such as diabetes, metabolic syndrome, obesity, cardiovascular disease, dyslipidemia and cancer. Current activation of AMPK which includes strenuous exercise, calorie restriction, pharmacological interventions mimetics metformin and resveratrol, and thiazolidinediones all have side effects which are detrimental or difficult to implement over time. Therefore, there exists a need in the market for an AMPK activator which has high bioavailability, low toxicity, and preferably is already a human metabolite which would lower overall pharmacological risk. The currently proposed invention meets those needs.